Vaccine compositions against pathogenic fungi and methods for use thereof

ABSTRACT

The present disclosure provides compositions comprising mutant fungi include an inactive form of the sterylglucosidase enzyme. The present disclosure is also directed to vaccine based compositions, which include a mutant fungus that prohibit pathogenic fungal infection. This disclosure also provides methods for administering these compositions as a prophylaxis against fungal infection, as well as methods for isolating sterylglucosides that include the use of such mutant fungal compositions.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority from U.S. ProvisionalApplication No. 62/372,894, filed Aug. 10, 2016, the entire contents ofwhich are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersAI56168 and R0171142 awarded by the National Institute of Health-NIAID.The government has certain rights in the invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The Sequence Listing in an ASCII text file, 33936_Seq_ST25.txt of 29 KB,created on Aug. 9, 2017, and submitted to the United States Patent andTrademark Office via EFS-Web, is incorporated herein by reference.

BRIEF DESCRIPTION

The present disclosure provides compositions comprising mutant fungithat lack the sterylglucosidase enzyme. This disclosure further providesvaccine compositions effective against pathogenic fungi, comprising amutant fungus lacking the sterylglucosidase enzyme, as well as methodsfor isolating sterylglucosides using the mutant fungus compositions ofthe instant application.

BACKGROUND

The human fungal pathogen Cryptococcus neoformans is an opportunisticfungal pathogen and the causative agent of the disease cryptococcosis.Cryptococcus neoformans is able to rapidly and effectively adapt tovarying conditions, favoring its survival in the environment and in theinfected host. Infections caused by Cryptococcus neoformans andCryptococcus gattii lead to more than 600,000 deaths per year (Park etal., 2009), especially among immunocompromised individuals. Cryptococcusneoformans is the leading cause of fungal meningitis worldwide. Inaddition to afflicting the central nervous system, Cryptococcusneoformans can cause significant damage to most major organ systemsincluding the heart, kidney and liver. Patients at particular risk arethose with HIV/AIDS, autoimmune disorders, long term steroid treatments,and patients undergoing solid organ or bone marrow transplantation.Importantly, some species of Cryptococcus gattii have been shown toinfect also immunocompetent subjects, causing mild to lethal pneumonia.

Many microbial phenotypes have been specifically correlated withvirulence in these opportunistic pathogens, such as capsule production,melanin formation, and the secretion of various proteins. Additionally,cellular features such as the cell wall and morphogenesis play importantroles in the interaction of Cryptococcus with host immune recognitionand response pathways.

Despite its significant public health burden, no vaccines currentlyexist in the clinic for cryptococcosis (or other fungal infectionsNanjappa and Klein, 2014). Although experimental vaccines have beendeveloped using the glucuronoxylomannan (GXM) capsule bound to tetanustoxoid (Devi et al., 1991; Casadevall et al., 1992; Devi, 1996), theseformulations have not been translated to the clinic and have sufferedfrom drawbacks such as inducing detrimental antibodies in mice(Casadevall and Pirofski, 2005; Datta and Pirofski, 2006). Recentattempts in the mouse models of cryptococcosis have been focused on theuse of genetically engineered C. neoformans strains that generatecytokines (Wormley et al., 2007; Wozniak et al., 2011) or proteinpreparations from C. gattii administered prior to infection (Chaturvediet al., 2014). Although these attempts have provided valuable insights,studies are still limited and shortcomings exist. For example, completeimmunity against C. gattii (responsible for severe infections in theUSA; Datta et al., 2009; Walraven et al., 2011) has not been achieved(Chaturvedi et al., 2014) demonstrating the need for the development ofmore effective vaccines against fungal infections.

SUMMARY OF THE DISCLOSURE

In one aspect, this disclosure provides a composition comprising amutant fungus, wherein said mutant fungus comprises an inactivatedSterylglucosidase (Sgl1) gene or homolog thereof.

Another aspect of this invention is directed towards a vaccinecomprising an effective amount of a mutant fungus comprising aninactivated Sterylglucosidase (Sgl1) gene homolog. In some embodiments,the effective amount for the mutant pathogenic fungi is between 1×10⁴fungal cells and 5×10⁵ fungal cells per administration. In a specificembodiment, the vaccine is an inactivated vaccine.

In some embodiments, the vaccine provides protection against pathogenicfungal infections. In a specific embodiment, pathogenic fungalinfections the vaccine protects against comprise infections by fungi ofgenus selected from the group consisting of Cryptococcus, Aspergillus,and Candida. In a specific embodiment, the pathogenic fungal infectionscomprise infections caused by dimorphic fungi selected from the groupconsisting of Coccidioides immitis, Paracoccidioides brasiliensis,Candida albicans, Ustilago maydis, Blastomyces dermatitidis, Histoplasmacapsulatum, Sporothrix schenckii, and Emmonsia sp.

Another aspect of this application provides a pharmaceutical compositioncomprising the vaccine of claim 7 and a pharmaceutically acceptablecarrier.

A different aspect of this disclosure provides a method for producingsterylglucosides comprising: providing a mutant fungus comprising aninactivated Sterylglucosidase (Sgl1) gene or homolog thereof; expressingsaid mutant fungus in a fungal cell, wherein said fungal cell producessterylglucosides; and isolating said sterylgucosides.

In some embodiments, the mutant fungus of this disclosure lacks theability to metabolize sterylglucosides (SGs). In some embodiments, themutant fungus accumulates sterol glycosides.

In some embodiments, the mutant fungus is avirulent.

In some embodiments, the fungus is from a Cryptococcus genus. In aspecific embodiment, said mutant fungus is selected from the groupconsisting of Cryptococcus neoformans, Cryptococcus gatii, Cryptococcusalbidus, Cryptococcus uniguttulatus, Candida albicans, Aspergillusfumigatus and other fungi in which the Sgl1 gene or homolog thereof isdeleted.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A-1E. CNAG_05607 enzyme has sterylglucosidase and notglucosylceramidase activity. (A) Cryptococcus neoformans CNAG_05607 (Cn5607) metabolizes plants SGs in a dose dependent manner and (B)metabolizes SGs extracted from Cn cells (Cn SGs). (C) pH dependence ofCn 5607 activity, as measured based on its ability to cleave plants SGsand produce free sterols, showed maximal activity at pH 4.5. Results arebased on the measurement of free sterols performed by liquidchromatography-mass spectrometry (LC-MS). Cn 5607 does not metabolize C.neoformans GlcCer (Cn GlcCer) as analyzed by (D) thin layerchromatography (TLC) or by (E) LC-MS (Cerezyme is an analog of the humanenzyme β-glucocerebrosidases, and was used as a positive control). Cn5607 is the endoglycoceramidase-related protein 2 (EGCrP2) alsoidentified by Watanabe et al. (2015). Empty V, empty vector.

FIG. 2A-2H. Deletion of SGL1 causes accumulation of SGs and not GlcCer.Analysis of sterylglucosides (SGs) and glucosylceramide (GlcCer) wasperformed by (A) TLC and (B-D) gas chromatography-(E-Gmass spectrometry.Results show that the Δsgl1 mutant dramatically accumulates SGs (A,B-D),which are normally undetectable in wild-type (WT) or reconstitutedstrain (Δsgl1+SGL1). In contrast, the level of GlcCer (A,E-G) in theΔsgl1 mutant is identical to the one observed in the WT or Δsgl1+SGL1reconstituted strain. Chromatograms are representatives of threeseparate experiments showing similar results. Peaks denote: (1)Dehydroergosteryl-β-D-glucoside*; (2) Ergosteryl-β-D-glucoside; (3)Ergosta-7,22-dien-3-oyl-β-D-glucoside*; (4) Fecosteryl-β-D-glucoside*;(5) Episteryl-β-D-glucoside*; (6) Lanosteryl-β-D-glucoside* (*: putativestructures). (H) Structure and electron-impact (EI) mass spectrum ofpeak number 2.

FIG. 3A-3C. Deletion of the SGL1 gene in C. neoformans abolishesvirulence. (A) Virulence studies showed that 100% of mice infected withΔsgl1 survived the infection whereas mice infected with C. neoformans WTH99 or with Δsgl1+SGL1 reconstituted strain succumbed to infectionwithin 24±6 and 21±7 days, respectively; n=8 mice in each group. (B)Lung tissue burden analysis showed that the Δsgl1 mutant is eliminatedfrom the lungs after 14 days of inoculation; n=3 mice each at timepoint. (C) Brain tissue burden analysis showed that Δsgl1 is not foundin the brain at anytime during the course of experiment; n=3 mice ateach time point.

FIG. 4A-4D. Vaccination studies. (A) Mice were “pre-treated” with Δsgl1and, after 30 days (time 0), challenged with a lethal dose of C.neoformans wild-type H99 (Cn WT) or Cryptococcus gattii WT R265 (Cg265). Mice exposed to Δsgl1 remained alive during the course ofexperiment (80 days post-infection) whereas all mice that were notexposed to Δsgl1 but to vehicle (PBS) or to the Δgcs1 mutant strain andthen challenged with Cn WT succumbed to infection within 35 days; n=8mice in each group. (B) Pre-treatment with Δsgl1 completely protectedCD4⁺ T-cell depleted mice from a subsequent lethal challenge with C.neoformans wild-type H99 (WT); n=8 mice in each group. Depletion of CD4⁺was achieved by administering anti-CD4 (Ab) weekly during the entirecourse of the experiment. The depletion was confirmed by flow cytometryperformed at the day of Cn challenge (C-D). Results are representativeof three separate experiments showing similar results.

FIG. 5. Alignment of the entire protein sequences of C. neoformansprotein EGCrP1 (SEQ ID NO: 7), C. neoformans protein Sgl1 (SEQ ID NO:9), S. cerevisiae protein Egh1 (SEQ ID NO: 8) and C. albicans proteinSgl1 ((SEQ ID NO: 10)). Identical conserved residues are shown in red,homologous residues in at least 3 of the sequences are shown in yellow.The protein homology scores were 45% for C. neoformans EGCrP1 (SEQ IDNO: 7 and GenBank Acc. No: BAL46040.1) and C. neoformans Sgl1 (SEQ IDNO: 9), 42% for C. neoformans EGCrP1 and S. cerevisiae Egh1 (SEQ ID NO:8, and GenBank Acc. No: DAA08553.1), and 45% for C. neoformans EGCrP1(SEQ ID NO: 7) and C. albicans putative Sgl1 (SEQ ID NO: 10).

FIG. 6. CNAG_05607 enzyme has cholesterol glucosidase activity. Therelease of cholesterol from cholesterol glycoside by CNAG_05607 enzymewas monitored using GC-MS. Empty vector and Cerezyme were used asnegative control. CG, Cholesterol glucoside; Empty V, empty vector.

FIG. 7A-7C. Deletion and reconstitution of the SGL1 gene. (A) Strategyfor the deletion of SGL1 in C. neoformans wild-type (WT) and creation ofthe mutant strain Δsgl1. (B) Strategy for the generation of thecomplemented strain Δsgl1+SGL1. (C) Southern Blot hybridization analysisof genomic DNA of WT, Δsgl1 and Δsgl1+SGL1 digested with ScaI and KpnIand screened with the “SGL1” probe (gray bar in B). Results show thatthe SGL1 gene has been deleted in the Δsgl1 mutant and reconstituted inthe Δsgl1+SGL1 strain. Gene deletion was confirmed using a second probe(5′ UTR—black bar in B). 5′ UTR, 5′ untranslated region; 3′ UTR, 3′untranslated region; NAT1, nourseothricin 1; Sgl1, sterylglucosidase 1;HYG. Hygromycin B.

FIG. 8A-8F. Histopathology of brains and lungs obtained from the CBA/Jmice infected intranasally with C. neoformans wild-type (WT), Δsgl1, andΔsgl1+SGL1 strains. The brains (A, C and E) were stained withmucicarmine stain, the lungs (B, D, and F) were stained with Hematoxylinand Eosin (H&E) stain. Black bar, 50 μm.

FIG. 9A-9J. Characterization of virulence phenotypes in the Δsgl1 mutantstrain. (A-B) C. neoformans wild-type (WT), Δsgl1, and Δsgl1+SGL1strains showed identical growth in DMEM at 37° C., 5% CO2 and at pH 7.4(A) or pH 4 (B). (C) C. neoformans cells were plated on mediumcontaining L-DOPA and the melanin produced by WT, Δsgl1, or Δsgl1+SGL1strain was similar. (D-F) Capule size of WT(D), Δsgl1 (E), or Δsgl1+SGL1(F) strains was similar when cells were incubated in DMEM at 37° C., 5%CO2. White bar, 10 μm (G) Cell body and capsule size of WT, Δsgl1, orΔsgl1+SGL1 strains, reported by measuring the cell body and capsule sizeof 50 cells for each strain under the microscope. (H) There were nomajor differences among WT, Δsgl1, and Δsgl1+SGL1 in their ability togrow under stressed conditions (hydrogen peroxide and nitrosativestress). (I-J) Phagocytosis (I) and intracellular killing (J) within amacrophage-like cell line J774A. 16 was identical between WT, Δsgl1, orΔsgl1+SGL1 strains at 2 and 24 hours, respectively.

FIG. 10. Isolation of sterolglucosides from Cryptococcus neoformansΔsgl1. Thin Layer Chromathography (TLC) of sterolglucosides isolatedfrom Cryptococcus neoformans and stained with orcinol. Lane 1,sterolglucosides standard from plants; Lane 2, glucosylceramide standardfrom soy; lane 3; purified sterolglucosides from C. neoformans.

DETAILED DESCRIPTION

It has been demonstrated herein that a non-pathogenic (avirulent) mutantstrain of pathogenic fungi that lacks the gene to metabolizesterylglucosides (SGs) can be used as a vaccine to protect a hostagainst infection by virulent strains of fungi such as Cryptococcusstrains (including but not limited to Cryptococcus neoformans,Cryptococcus gatii, Cryptococcus albidus, and Cryptococcusuniguttulatus), Aspergillus nidulans, Candida albicans, and otherpathogenic dimorphic fungi (including but not limited to Coccidioidesimmitis, Paracoccidioides brasiliensis, Candida albicans, Ustilagomaydis, Blastomyces dermatitidis, Histoplasma capsulatum, Sporothrixschenckii, and Emmonsia sp.). Accordingly, the present disclosure isdirected to compositions and vaccines comprising a mutant funguscomprising an inactivated Sterylglucosidase (Sgl1) gene homolog (aka.delta SGL1 (Δsgl1)).

A “homolog” means a gene related to a second gene by descent from acommon ancestral DNA sequence, therefore, the correspondingpolynucleotide/polypeptide has a certain degree of homology, that is tosay sequence identity (preferably at least 40%, more preferably at least60%, even more preferably at least 65%, particularly preferred at least66%, 68%, 70%, 75%, 80%, 86%, 88%, 90%, 92%, 95%, 97% or 99%).“Sterylglucosidase (Sgl1) gene homolog” furthermore means that thefunction is equivalent to the function of the Sterylglucosidase (Sgl1)gene.

The Sterylglucosidase (Sgl1) gene encodes for an enzyme that does notmetabolizes long-chain Δ8-C9 methyl glucosylceramides in Cryptococcusneoformans. The Sgl1 enzyme only metabolizes short-chainglucosylceramides (e.g. C6-glucosylceramide), which are notphysiologically relevant because these species of glucosylceramides arenot synthesized in fungal cells. Sterylglucosidase (Sgl1) gene homologsencode an enzyme with sterylglucosidase activity (i.e., the ability tometabolize sterolglycosides). BLAST database searches with either thenucleotide sequence of Cryptococcus neoformans Sterylglucosidase (Sgl1)gene or with the amino acid sequence of the Sgl1 gene provide otherhomolog genes and proteins in other fungi species. For example, a BLASTsearch of CNAG_05607 in Saccharomyces genome database reveals a homologgene called YIR007W in Saccharomyces cerevisiae. In addition, proteinalignments reveals similarities between Sgl1 homologs from differentspecies (FIG. 5). Protein homology scores was 45% for C. neoformansEGCrP1 and C. neoformans Sgl1, 42% for C. neoformans EGCrP1 and S.cerevisiae Egh1, and 45% for C. neoformans EGCrP1 and C. albicansputative Sgl1.

In some embodiments, the inactivation of the Sterylglucosidase (Sgl1)gene includes a deletion of the whole or a part of the gene such that nofunctional protein product is expressed (also known as gene knock out).The inactivation of a gene may include a deletion of the promoter or thecoding region, in whole or in part, such that no functional proteinproduct is expressed. In other embodiments, the inactivation ofSterylglucosidase (Sgl1) includes introducing an inactivating mutationto the gene, such as an early STOP codon in the coding sequence of thegene, such that no functional protein product is expressed. Deletion orinactivation of the Sterylglucosidase (Sgl1) gene leads to significantaccumulation of sterol-glucosides in cells. Sterolglucosides aremolecules which are undetectable in wild-type fungi strains.Sterolglucosides accumulated by the mutant fungi trigger host immuneresponses against pathogenic fungi and help protect from, and preventagainst fungal infections.

In some embodiments, gene inactivation is achieved using available genetargeting technologies in the art. Examples of gene targetingtechnologies include the Cre/Lox system (described in Kühn, R., & M.Torres, R., Transgenesis Techniques: Principles and Protocols, (2002),175-204), homologous recombination (described in Capecchi, Mario R.Science (1989), 244: 1288-1292), TALENs (described in Sommer et al.,Chromosome Research (2015), 23: 43-55, and Cermak et al., Nucleic AcidsResearch (2011): gkr218), and CRISPR Cas system as described in Ran F Aet al., Nature Protocols (2013).

In one embodiment, Sterylglucosidase (Sgl1) inactivation is achieved bybiolistic transformation, which is a gene targeting systems well knownin the art with reagents and protocols readily available (Toffaletti etal., Journal of Bacteriology, 175.5 (1993): 1405-1411).

Inactivation of the Sterylglucosidase (Sgl1) gene also causes the fungusto become avirulent (non-pathogenic).

As used herein, “pathogenic” means causing symptoms of a diseaseassociated with the pathogen. As used herein “a non-pathogenic strain”or “an avirulent strain” is a strain of microorganism which has theability to colonize and replicate in an infected individual, but whichdoes not cause disease symptoms associated with virulent strains of thesame species of microorganism. The microbe may belong to a genus or evena species that is normally pathogenic but must belong to a strain thatis avirulent. Avirulent strains are incapable of inducing a full suiteof symptoms of the disease that is normally associated with its virulentpathogenic counterpart. Avirulent strains of microorganisms may bederived from virulent strains by mutation.

In some embodiments, the mutant fungus is from the Cryptococcus genus.In other embodiments, the mutant fungus can be from any one of thefollowing Cryptococcus fungal strains: Cryptococcus neoformans,Cryptococcus gatii, Cryptococcus albidus, and Cryptococcusuniguttulatus.

In one embodiment, the mutant fungus comprises an inactivatedSterylglucosidase (Sgl1) gene homolog that can be included in apharmaceutical composition. As used herein, a pharmaceutical compositionis a formulation which contains at least one active ingredient as wellas, for example, one or more excipients, buffers, carriers, stabilizers,preservatives and/or bulking agents, and is suitable for administrationto a patient to achieve a desired effect or result. The pharmaceuticalcompositions disclosed herein can have diagnostic, preventative (i.e.,phrophylactic), cosmetic and/or research utility in various species,such as for example in human patients or subjects.

This disclosure also provides a composition comprising a combination asdescribed above and a pharmaceutically acceptable carrier. For thepurposes of this disclosure, “pharmaceutically acceptable carriers”means any of the standard pharmaceutical carriers. Examples of suitablecarriers are well known in the art and may include, but are not limitedto, any of the standard pharmaceutical carriers such as a phosphatebuffered saline solution and various wetting agents. Other carriers mayinclude additives used in tablets, granules and capsules, and the like.Typically such carriers contain excipients such as starch, milk, sugar,certain types of clay, gelatin, stearic acid or salts thereof, magnesiumor calcium stearate, talc, vegetable fats or oils, gum, glycols or otherknown excipients. Such carriers may also include flavor and coloradditives or other ingredients. Compositions comprising such carriersare formulated by well-known conventional methods.

The pharmaceutical preparations of the present disclosure can be made upin any conventional form including, inter alia: (a) a solid form fororal administration such as tablets, capsules (e.g. hard or soft gelatincapsules), pills, cachets, powders, granules, and the like; (b)preparations for topical administrations such as solutions, suspensions,ointments, creams, gels, micronized powders, sprays, aerosols and thelike. The pharmaceutical preparations may be sterilized and/or maycontain adjuvants such as preservatives, stabilizers, wetting agents,emulsifiers, salts for varying the osmotic pressure and/or buffers.

The pharmaceutical compositions of the present disclosure can be used inliquid, solid, tablet, capsule, pill, ointment, cream, nebulized orother forms as explained below. In some embodiments, the composition ofthe present disclosure can be administered by different routes ofadministration such as oral, oronasal, parenteral or topical.

“Oral” or “peroral” administration refers to the introduction of asubstance, such as a vaccine, into a subject's body through or by way ofthe mouth and involves swallowing or transport through the oral mucosa(e.g., sublingual or buccal absorption) or both.

“Oronasal” administration refers to the introduction of a substance,such as a vaccine, into a subject's body through or by way of the noseand the mouth, as would occur, for example, by placing one or moredroplets in the nose. Oronasal administration involves transportprocesses associated with oral and intranasal administration.

“Parenteral administration” refers to the introduction of a substance,such as a vaccine, into a subject's body through or by way of a routethat does not include the digestive tract. Parenteral administrationincludes subcutaneous administration, intramuscular administration,transcutaneous administration, intradermal administration,intraperitoneal administration, intraocular administration, andintravenous administration. For the purposes of this disclosure,parenteral administration excludes administration routes that primarilyinvolve transport of the substance through mucosal tissue in the mouth,nose, trachea, and lungs.

Formulations suitable for parenteral administration comprise acomposition comprising a mutant fungus comprising an inactivatedSterylglucosidase (Sgl1) gene homolog in combination with one or morepharmaceutically-acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacterostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the formulations suitable for parenteral administrationinclude water, ethanol, polyols (e.g., such as glycerol, propyleneglycol, polyethylene glycol, and the like), and suitable mixturesthereof, vegetable oils, such as olive oil, and injectable organicesters, such as ethyl oleate. Proper fluidity can be maintained, forexample, by the use of coating materials, such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

Formulations suitable for parenteral administration may also containadjuvants such as preservatives, wetting agents, emulsifying agents anddispersing agents. Prevention of the action of microorganisms may beensured by the inclusion of various antibacterial and antifungal agents,for example, paraben, chlorobutanol, phenol sorbic acid, and the like.It may also be desirable to include isotonic agents, such as sugars,sodium chloride, and the like into the compositions. In addition,prolonged absorption of the injectable pharmaceutical form may bebrought about by the inclusion of agents which delay absorption such asaluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a compositioncomprising a mutant fungus comprising an inactivated Sterylglucosidase(Sgl1) gene homolog, it is desirable to slow the absorption of the drugfrom subcutaneous or intramuscular injection. This may be accomplishedby the use of a liquid suspension of crystalline or amorphous materialhaving poor water solubility. The rate of absorption of the drug thendepends upon its rate of dissolution which, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally-administered formulation is accomplished by dissolving orsuspending the composition comprising a mutant fungus comprising aninactivated Sterylglucosidase (Sgl1) gene homolog in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of acomposition comprising a mutant fungus comprising an inactivatedSterylglucosidase (Sgl1) gene homolog or in biodegradable polymers suchas polylactide-polyglycolide. Depending on the ratio of the compositioncomprising a mutant fungus comprising an inactivated Sterylglucosidase(Sgl1) gene homolog to polymer, and the nature of the particular polymeremployed, the rate of drug release can be controlled. Examples of otherbiodegradable polymers include poly (orthoesters) and poly (anhydrides).Depot injectable formulations are also prepared by entrapping acomposition comprising a mutant fungus comprising an inactivatedSterylglucosidase (Sgl1) gene homolog in liposomes or microemulsionswhich are compatible with body tissue.

“Topical administration” means the direct contact of a substance withtissue, such as skin or membrane, particularly the oral or buccalmucosa.

For topical administration to the skin or mucous membrane theaforementioned composition is preferably prepared as ointments,tinctures, creams, gels, solution, lotions, sprays; aerosols and drypowder for inhalation, suspensions, shampoos, hair soaps, perfumes andthe like. In fact, any conventional composition can be utilized in thisinvention. Among the preferred methods of applying the compositioncontaining the agents of this invention is in the form of an ointment,gel, cream, lotion, spray; aerosol or dry powder for inhalation. Thepharmaceutical preparation for topical administration to the skin can beprepared by mixing the aforementioned active ingredient with non-toxic,therapeutically inert, solid or liquid carriers customarily used in suchpreparation. These preparations generally contain 0.01 to 5.0 percent byweight, or 0.1 to 1.0 percent by weight, of the active ingredient, basedon the total weight of the composition.

In preparing the topical preparations described above, additives such aspreservatives, thickeners, perfumes and the like conventional in the artof pharmaceutical compounding of topical preparation can be used. Inaddition, conventional antioxidants or mixtures of conventionalantioxidants can be incorporated into the topical preparationscontaining the aforementioned active agent. Among the conventionalantioxidants which can be utilized in these preparations are includedN-methyl-a-tocopherolamine, tocopherols, butylated hydroxyanisole,butylated hydroxytoluene, ethoxyquin and the like.

Cream-based pharmaceutical formulations containing the active agent,used in accordance with this invention, are composed of aqueousemulsions containing a fatty acid alcohol, semi-solid petroleumhydrocarbon, ethylene glycol and an emulsifying agent.

Ointment formulations containing the active agent in accordance withthis invention comprise admixtures of a semi-solid petroleum hydrocarbonwith a solvent dispersion of the active material. Cream compositionscontaining the active ingredient for use in this invention preferablycomprise emulsions formed from a water phase of a humectant, a viscositystabilizer and water, an oil phase of a fatty acid alcohol, a semi-solidpetroleum hydrocarbon and an emulsifying agent and a phase containingthe active agent dispersed in an aqueous stabilizer-buffer solution.Stabilizers may be added to the topical preparation. Any conventionalstabilizer can be utilized in accordance with this invention. In the oilphase, fatty acid alcohol components function as a stabilizer. Thesefatty acid alcohol components function as a stabilizer. These fatty acidalcohol components are derived from the reduction of a long-chainsaturated fatty acid containing at least-14 carbon atoms. The ointments,pastes, creams and gels may contain, in addition to a mutant funguscomprising an inactivated Sterylglucosidase (Sgl1) gene homolog,excipients, such as animal and vegetable fats, oils, waxes, paraffins,starch, tragacanth, cellulose derivatives, polyethylene glycols,silicones, bentonites, silicic acid, talc and zinc oxide, or mixturesthereof. Powders and sprays can contain, in addition to a compositioncomprising a mutant fungus comprising an inactivated Sterylglucosidase(Sgl1) gene homolog, excipients such as lactose, talc, silicic acid,aluminum hydroxide, calcium silicates and polyamide powder, or mixturesof these substances. Sprays can additionally contain customarypropellants, such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane. Also, conventional perfumesand lotions generally utilized in topical preparation for the hair canbe utilized in accordance with this invention. Furthermore, if desired,conventional emulsifying agents can be utilized in the topicalpreparations of this invention.

An oral dosage form comprises cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, or capsules of hard or soft gelatin, methylcellulose or of anothersuitable material easily dissolved in the digestive tract, eachcontaining a predetermined amount of a composition comprising a mutantfungus comprising an inactivated Sterylglucosidase (Sgl1) gene homologas an active ingredient. Each tablet, pill, sachet or capsule canpreferably contain from about 1×10¹ to about 1×10⁹ cells of mutant fungias active ingredient. The oral dosages contemplated in accordance withthe present invention will vary in accordance with the needs of theindividual patient as determined by the prescribing physician.

A compound may also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (e.g., capsules, tablets,pills, dragees, powders, granules and the like), a compositioncomprising a mutant fungus comprising an inactivated Sterylglucosidase(Sgl1) gene homolog is mixed with one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate, (5) solution retarding agents,such as paraffin, (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, acetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered peptide orpeptidomimetic moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of a composition comprising a mutantfungus comprising an inactivated Sterylglucosidase (Sgl1) gene homologtherein using, for example, hydroxypropylmethyl cellulose in varyingproportions to provide the desired release profile, other polymermatrices, liposomes and/or microspheres. They may be sterilized by, forexample, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved in sterile water, or some othersterile injectable medium immediately before use. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the composition comprising a mutant fungus comprisingan inactivated Sterylglucosidase (Sgl1) gene homolog only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes. Thecomposition comprising a mutant fungus comprising an inactivatedSterylglucosidase (Sgl1) gene homolog can also be in micro-encapsulatedform, if appropriate, with one or more of the above-describedexcipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to a composition comprising a mutant funguscomprising an inactivated Sterylglucosidase (Sgl1) gene homolog, theliquid dosage forms may contain inert diluents commonly used in the art,such as, for example, water or other solvents, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Besides inert diluents, the oral compositions can also includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to a composition comprising a mutant funguscomprising an inactivated Sterylglucosidase (Sgl1) gene homolog, maycontain suspending agents as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more compositionscomprising a mutant fungus comprising an inactivated Sterylglucosidase(Sgl1) gene homolog with one or more suitable nonirritating excipientsor carriers comprising, for example, cocoa butter, polyethylene glycol,a suppository wax or a salicylate, and which is solid at roomtemperature, but liquid at body temperature and, therefore, will melt inthe rectum or vaginal cavity and release the active agent. Formulationswhich are suitable for vaginal administration also include pessaries,tampons, creams, gels, pastes, foams or spray formulations containingsuch carriers as are known in the art to be appropriate.

Compositions comprising a mutant fungus comprising an inactivatedSterylglucosidase (Sgl1) gene homolog can be alternatively administeredby aerosol. For example, this can be accomplished by preparing anaqueous aerosol, liposomal preparation or solid particles containing acomposition comprising a mutant fungus comprising an inactivatedSterylglucosidase (Sgl1) gene homolog preparation. A nonaqueous (e.g.,fluorocarbon propellant) suspension could be used. Sonic nebulizers canalso be used. An aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants, innocuous proteins like serumalbumin, sorbitan esters, oleic acid, lecithin, amino acids such asglycine, buffers, salts, sugars or sugar alcohols. Aerosols generallyare prepared from isotonic solutions.

In another embodiment, said pharmaceutical composition comprising themutant fungus comprising an inactivated Sterylglucosidase (Sgl1) genehomolog is included in a vaccine composition. Said vaccine compositionmay be administered by any one of the routes of administration describedabove.

In one embodiment, the vaccine composition of the present disclosure isan attenuated vaccine comprising a mutant fungus comprising aninactivated Sterylglucosidase (Sgl1) gene homolog (Δsgl1). An attenuatedvaccine is a vaccine created by reducing the virulence of a pathogen,but still keeping it viable (or “live”). Attenuation takes an infectiousagent and alters it so that it becomes harmless or less virulent.

In another embodiment the vaccine composition of the present disclosureis an inactivated vaccine comprising a mutant fungus comprising aninactivated Sterylglucosidase (Sgl1) gene homolog (delta Sgl1). Aninactivated vaccine (or killed vaccine) is a vaccine consisting of virusparticles, bacteria, or other pathogens that have been grown in cultureand then killed using a method such as heat or formaldehyde. Incontrast, live vaccines (which are nearly always attenuated vaccines)use pathogens that are still alive (but are almost always attenuated,that is, weakened). Pathogens for inactivated vaccines are grown undercontrolled conditions and are killed as a means to reduce infectivity(virulence) and thus prevent infection from the vaccine.

In yet another embodiment, the vaccine composition may further comprisean appropriate adjuvant. Appropriate adjuvants include but are notlimited to aluminum hydroxide, aluminum phosphate, gamma inulin,algammulin (a combination of aluminum hydroxide and gamma inulin),cholecalciferol in oil, an oil in water emulsion OWEM1, containingsqualene, tween-80, Span-85 in 10 mM phosphate-citrate buffer, oil inwater emulsion OWEM2 containing squalene, tween-80, Span-85, alphatocopherol in phosphate-citrate buffer, and saponin. The saponin-basedadjuvants include but are not limited to QS21, QuilA, tomatine, ISCOMs,ISCOMATRIX and GPI-0100. In an embodiment, the range of QS21 is fromabout 25 to 200 μg. In another embodiment, the QS21 is about 100 μg. Ina separate embodiment, the adjuvant is GPI-0100 and the range is fromabout 1 to 25 mg. In yet another embodiment, GPI-0100 is about 10 mg.

In one embodiment of the disclosure, the vaccine composition isdelivered to a subject in a pharmaceutically effective dose. The term“pharmaceutically effective dose” as used herein refers to the amount ofthe vaccine preparation, which is effective for producing a desiredvaccinal effect. As is known in the art of pharmacology, the preciseamount of the pharmaceutically effective dose of a vaccine preparationthat will yield the most effective results in terms of efficacy ofadministration of the composition in a given subject will depend upon,for example, the activity, the particular nature, pharmacokinetics,pharmacodynamics, and bioavailability of a particular vaccinepreparation, physiological condition of the subject (including race,age, sex, weight, diet, disease type and stage, general physicalcondition, responsiveness to a given dosage and type of medication), thenature of pharmaceutically acceptable carriers in a formulation, theroute and frequency of administration being used, to name a few.However, the above guidelines can be used as the basis for fine-tuningthe treatment, e.g., determining the optimum dose of administration,which will require no more than routine experimentation consisting ofmonitoring the subject and adjusting the dosage. Remington: The Scienceand Practice of Pharmacy (Gennaro ed. 20th edition, Williams & WilkinsPa., USA (2000)).

In one embodiment, the vaccine comprises about 1×10¹, 1×10², 1×10³,1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹ or more mutant fungi cells peradministration dose.

In one embodiment, an SGL1 deletion mutant of a fungus (delta SGL1) isused for purification of sterylglucosides. In another embodiment, themutant fungus is from Cryptococcus genus. In yet another embodiment, theCryptococcus fungus is selected from the group consisting ofCryptococcus neoformans, Cryptococcus gatii, Cryptococcus albidus, andCryptococcus uniguttulatus. Here, it has been discovered that when theSGL1 gene homolog is deleted in these fungi, sterylglucosides (SGs)begin to accumulate. SGs can be isolated with known biochemical methodsin the art.

In another aspect of the present disclosure, mutant fungi exhibiting aninactivated sterylglucosidase enzyme can be used to producesterylglucoside(s). Specifically, fungal sterylglucosides. Here, amutant fungi composition including an inactivated SGL1 gene or homologthereof can be provided and expressed in a cell or collection of cellssuch as, for example, bacterial cell, fungal cell, yeast cell, or othercell known by those of ordinary skill in the art to be used in proteinproduction. In certain embodiments, the mutant fungi composition isexpressed in cells by transfection of the cells with a plasmid or vectorthat includes the mutant fungi comprising an inactivated SGL1 gene orhomolog thereof. The cells can then be grown for a period of timesufficient to express sterolglucoside(s) due to the inactivity of thesterolglucosidase exhibited by the mutant fungi composition. Forexample, the cells can be grown for at least 1 hour. In otherembodiments, the cells can be grown for at least 24 for ours. In anotherembodiment the cells can be grown for period of time from 1 to 72 hoursor greater. In other embodiments, the cells can be grown until adetectable amount of fungal sterolglucoside is detected in the cells.

In a specific embodiment, the cell expressing the mutant fungi is afungal cell or fungal strain. Non-limiting examples of such fungal cellsinclude Cryptococcus neoformans, Cryptococcus gatii, Cryptococcusalbidus, Cryptococcus uniguttulatus, Aspergillus nidulans, Candidaalbicans. In other embodiments the fungal cells are Coccidioidesimmitis, Paracoccidioides brasiliensis, Candida albicans, Ustilagomaydis, Blastomyces dermatitidis, Histoplasma capsulatum, Sporothrixschenckii, or Emmonsia sp.

The expression of the mutant fungi of the present disclosure leads tosignificant accumulation of sterolglucosides in the cells, which canthen be isolated and purified, as defined in further detail herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one skilled in the artto which this invention belongs. Although any methods and materialssimilar or equivalent to those described herein can also be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

The specific examples listed below are only illustrative and by no meanslimiting.

EXAMPLES Materials and Methods Strains, Plasmid, and Culture Conditions

The fungal strains used in this study were C. neoformans (Cn) var.grubii strain H99 wild-type (WT) and C. gattii strain R265 andSaccharomyces cerevisiae ΔYIR007W mutant (ScΔYIR) derived from BY4741.Bacterial strain Escherichia coli DH5-α (Life Technologies, Carlsbad,Calif., USA) was used as competent cells. The plasmid pCR II-TOPO 4.0 kbwas used for cloning, and pYES2/CT was used for expression of Cn 5607 inScΔYIR.

Bacterial strains were grown at 37° C. in Luria-Bertani medium with 75mg/L of ampicillin. Cn strains were routinely grown in YPD broth at 30°C. and 0.04% atmospheric CO₂ for 20-22 h with shaking at 250 rpm.Dulbecco's modified eagle media (DMEM) buffered with 25 mM HEPES and 2%glucose at pH 7.4 or pH 4 was used for growing Cn at 37° C. and 5% CO₂(i.e., physiologically relevant conditions). ScΔYIR strain transformedwith pYES2/CT or pYES2/CT− Cn 5607, was grown in yeast nitrogen base(YNB) without amino acids, 1.2 g/L amino acid mixture lacking uracil(ura-), and 2% glucose or 2% galactose (48 h) at 30° C. to induce Cn5607 expression.

Expression of Cn 5607 in S. cerevisiae

Cn 5607 was identified by blasting the Cn WT endoglycoceramidase-relatedProtein 1 (EGCrP1) sequence in Cn WT Broad Institute genome database[http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans/MultiHome.html].Two sequences were found: one 100% identical to EGCrP1 and another onewith an E-value of 9e-57, located on chromosome 14 (Cn 5607). To expressCn 5607 in S. cerevisiae strains, total RNA was extracted from Cn andthe cDNA was synthesized from 1 μg of the total RNA using SuperScriptIII RNase H-Reverse Transcriptase (Life Technologies). PCR was performedusing the cDNA as a template and the following expression primers:PRSGL1-5′ forward (5′-GAGCTCATGCCTCCTCCACCAGAAGT-3′) (SEQ ID NO: 1) andPRSGL1-5′ reverse (5′-TCTAGAAGCAATAACGCATTCAGGACA-3′) (SEQ ID NO: 2)carrying the restriction enzyme sites for SacI and XbaI respectively. A2,556-pb fragment was cloned into pCR II-TOPO vector generating plasmidpCR-Cn 5607 and sequenced. After digestion with SacI and XbaI, Cn 5607was inserted into pYES2/CT vector generating pYES2/CT-Cn 5607. ScΔYIRwas grown in YNB medium overnight at 30° C. and transformed withpYES2/CT empty-vector or pYES2/CT-Cn 5607 using lithium acetatetransformation, as previously described (Kawai et al., 2010). Aftertransformation, cells were plated on YNB ura-plates and incubated for2-3 days in 30° C. incubator. Then, S. cerevisiae colonies were patchedwith sterile toothpicks to fresh YNB ura-plates. To verify theexpression of Cn 5607, one single colony containing pYES2/CT orpYES2/CT-Cn 5607 construct was inoculated into 10 ml of YNBura-containing 2% of glucose and was grown overnight at 30° C. withshaking. The cells were washed twice with PBS 1× and a suitable amountof overnight culture necessary to obtain an OD₆₀₀ of 0.4 was transferredto a fresh YNB ura-medium containing 2% of galactose (induction medium)and incubated for 48 h at 30° C. with shaking. After 48 h, the cellswere washed and harvested by centrifugation at 3000 g for 5 min at 4°C., and the cell pellets was stored at −80° C. until ready to be used.

Total proteins were extracted from S. cerevisiae strains as previouslydescribed in Singh et al., 2011, the entire contents of which isexpressly incorporated herein by reference. Protein content was assessedby the method of Bradford using bovine serum albumin (Sigma-Aldrich, St.Louis, Mo., USA) as a standard. Fifty micrograms of S. cerevisiaeproteins were loaded onto SDS-PAGE and stained with Coomassie BrilliantBlue. Western Blot was used to detect the expression of recombinantfusion protein using Anti-His (C-term)-HRP antibody.

In Vitro Activity Assay of Cn 5607 Using Standard Plants SGs andPurified Cn SGs

The in vitro Cn 5607 enzymatic assay was performed using 20 μg ofstandard SGs (purified from plants and commercially available) or 10 μgof endogenously purified SGs as substrate and ScΔYIR+empty vector orScΔYIR-Cn 5607 as source of enzyme. The decrease in the intensity of theSGs band was monitored by thin layer chromatography (TLC). Plants SGswere incubated with 200 μg of ScΔYIR+empty vector or 50, 100, and 200 μgof ScΔYIR-Cn 5607 at 30° C. for 1 h. Endogenously purified Cn SGs wereincubated with 200 μg of ScΔYIR+empty vector or 100 μg of ScΔYIR-Cn 5607at 30° C. for 1 h. The reactions were terminated by the addition of 300μl of CHCl₃/MeOH (1:1 ratio) the lower phases were dried down,resuspended in 50 μl of CHCl₃/MeOH (2:1 ratio) and analyzed by TLC. Cn5607 specifically cleaves SGs, thus Cn 5607 was renamed asSterylglucosidase1 (Sgl1).

The pH dependence of Sgl1 was determined using as substrate SGs in a pHrange of 4.5-8 using the following buffers at the final concentration of50 mM: sodium acetate (pH 4.5-5.0), MES (pH 5.5-6.0), sodium phosphate(pH 6.5-7.0), and HEPES (pH 7.5-8.0). The optimal temperature of Sgl1was determined in the range from 25 to 37° C. The effect of detergentswas assessed using Triton X-100, Sodium Deoxycholate and CHAPS at theconcentration of 0.05, 0.15, and 0.3%.

In Vitro Activity Assay of Cn 5607 Using NBD-C₆-Glucosylceramide and CnLone Chain GlcCer

To verify the enzymatic activity of Cn 5607, different substrates wereused: NBD-C₆-glucosylceramide (NBD-C₆-GlcCer; Matreya, LLC,State-College, Pa., USA) and Cn long chain GlcCer. Briefly, 200 μg ofyeast proteins from ScΔYIR+empty vector or ScΔYIR-Cn 5607 were incubatedfirst with 20 μM of NBD-C₆-Glucosylceramide and at 30° C. for 1 h in afinal reaction volume of 100 μl. The production of NBD-C₆-Ceramide wasidentified as a fluorescent band using a PhosphorImager™ 860 STORM unitand ImageQuant analysis (GE Healthcare, Rahway, N.J., USA) as previouslydescribed (Rittershaus et al., 2006).

The in vitro Cn 5607 activity was also valuated using 10 μg of Cn longchain GlcCer and 200 μg of ScΔYIR+empty vector or ScΔYIR-Cn 5607 cellextracts. Cerezyme (10 μg), generously provided by the GenzymeCorporation (Cambridge, Mass., USA), was used as positive control forthe catalytic reaction. The reactions were terminated by the addition of300 μl of CHCl₃/MeOH (1:1 ratio), the samples were mixed and the phaseswere separated by centrifugation at 3000 g for 5 min. The lower phaseswere dried down using a SPD 2010 SpeedVac vacuum dryer (Thermo ElectronCorp.). The dried samples were resuspended in 50 μl of CHCl₃/MeOH (2:1ratio) and analyzed by TLC on silica gel plate (EMD Millipore,Billerica, Mass., USA) developed with chloroform/methanol/water(65:25:4, v/v/v) and stained with iodine and resorcinol. The in vitroactivity assay using Cn long chain GlcCer and ScΔYIR+empty vector orScΔYIR-Cn 5607 was also repeated with a longer incubation time (4 h) andthe results were evaluated by liquid chromatography-mass spectrometry(LC-MS).

Disruption and Reconstitution of SGL1 Gene in Cn

The SGL1 gene (locus number CNAG_05607 in C. neoformans var. grubiiserotype A genome database) was deleted using NAT1 (NourseothricinAcetyl transferase1) split marker. A knockout cassette was generatedcontaining a 1.035 bp of the 5′ untranslated region (5′UTR) upstream ofthe ATG start codon of the SGL1 gene and a 1.059 bp of the 3′UTR. The5′UTR was amplified by PCR using H99 genomic DNA as a template and thefollowing primers: 5′UTR-F (5′-GTCAAGCTAAGAGCTCCATTTGATCAGCGGGATTCT-3′)(SEQ ID NO: 3) and 5′UTR-R (5′-TCCACTCCGAACTAGTATCGCGTAAACGAAGAGGTG-3′)(SEQ ID NO: 4), containing SacI and SpeI sites, respectively(underlined). The 3′UTR was amplified by PCR using H99 genomic DNA as atemplate and the following primers: 3′UTR-F(5′-GTCAAGCTAATCTAGAAGCCCATTCTGGTTGTTCTG-3′) (SEQ ID NO: 5) and 3′UTR-R(5′-ACATCACACTTCTAGATTTAGCGAGCCACGTTTCT-3′) (SEQ ID NO: 6). Theamplified fragments were cloned in pCR II-TOPO and sequenced, generatingplasmid pCR-5′UTR-TOPO and pCR-3′UTR-TOPO. NAT1 gene, which confersresistance to the antibiotic nourseothricin (Werner BioAgents, Jena,Germany), under the control of Cn actin promoter was digested from theplasmid pCR-NAT1-TOPO by SacI and SpeI and ligated with 5′UTR digestedwith the same restriction enzyme generating pCR-5′UTR-NAT1-TOPO.Finally, pCR-5′UTR-NAT-TOPO was digested by EcoRV and ligated with 3′UTRgenerating the disruption cassette pCR-5′UTR-NAT1-3′UTR-TOPO that wasnamed pΔsgl1. The deletion scheme is illustrated in FIG. 7A. Cn WT wastransformed with the plasmid pΔsgl1 using biolistic DNA delivery device,as described previously (Toffaletti et al., 1993). Stable transformantswere grown on YPD plates containing 100 μg/ml of nourseothricin.Colonies were chosen randomly and genomic DNA was isolated and digestedwith EcoRV and KpnI for Southern blot analysis. The DNA fragments werescreened by probing with a fragment of 5′UTR. Transformant #106 showingdeletion of the SGL1 gene by insertion of the NAT1 was chosen anddesignated Δsgl1 mutant strain. SGL1 gene was reintroduced back into theΔsgl1 using the reconstitution cassette pSK-SGL1-HYG, which had theHygromycin B allele as selectable marker. The reconstitution scheme isillustrated in FIG. 7B. The plasmid pSK-SGL1-HYG was biolisticallydelivered into Δsgl1. Homologous recombinants were screened by Southernhybridization using a 800 bp fragment of the SGL1 open reading frame asprobes. Transformant #21 showing reconstitution of SGL1 gene wasdesignated Δsgl1+SGL1 reconstituted strain.

Wild-type, mutant, and reconstituted strains were characterized fortheir growth profile, capsule formation, stress response, andintracellular growth. For growth profile studies, WT, Δsgl1, andΔsgl1+SGL1 reconstituted strains were grown overnight in YPD at 30° C.,the cells were washed three times with PBS, counted, and diluted to afinal density of 10⁴ cells/ml in DMEM at pH 7.4 or pH 4 and incubated at37° C. in the presence of 5% CO₂. Aliquots were taken at different timepoints, diluted, and plated in duplicates onto YPD agar plates forassessment of CFUs. Capsule thickness and melanin production weredetermined as previously described (Wang et al., 1995; Shea et al.,2006). For oxidative stress studies, strains were spotted in serialdilution (10, 10⁶, 10⁵, 10⁴, 10³) on YPD agar plates with 25 mM HEPES(pH 7 or pH 4) supplemented with 5 mM H₂O₂, cells growth was assessedafter incubation at 30° C. for 96 h. Nitrosative stress response wasstudied by spotting the strains in serial dilution (10⁷, 10⁶, 10⁵, 10⁴,10³) on YNB agar plates with 25 mM succinate acid (pH 4) supplementedwith 0.1 mM NaNO₂. Cell growth was assessed after 96 h of incubation at30, 37° C. in atmospheric environment or 37° C. in the presence of 5%CO₂.

Phagocytosis and intracellular killing studies were performed in J774.16macrophage-like cells as previously described (Tripathi et al., 2012).Briefly, for phagocytosis experiments cells were plated in a 96 wellplate in Dulbecco's minimal essential medium (DMEM) supplemented with10% fetal bovine serum (FBS). C. neoformans cells were grown overnightin YPD at 30° C. Cells were washed twice in PBS and counted.Approximately 10⁵ cells in DMEM+FBS medium were opsonized with 10 μg/mlof anti-GXM monoclonal antibody 18B7 (kindly provided by Dr. ArturoCasadevall) and added to macrophage-like cells activated with 50units/ml of recombinant murine gamma interferon and 0.3 μg/ml oflipopolysaccharide at an effector-to-target ratio of 1:1. Afterincubation for 2 h, extracellular C. neoformans cells were washed withthree changes of warm DMEM medium and fresh medium. Then, 200 μl ofsterile water was added to each well and the macrophage-like cells werelysed by pipetting several times. CFUs were analyzed by plating them onYPD agar plates and the numbers of internalized fungal cells werereported. Intracellular killing were performed in the same way with thefollowing change: extracellular C. neoformans cells were washed off once2 h after the initial incubation and another time after 24 h ofincubation. Macrophage-like cells were lysed after 24 h by pipettingseveral times and CFUs were analyzed by plating them on YPD agar plates.

Lipid Analysis of Cryptococcus Strains by TLC

Total lipids from Cryptococcus strains were extracted, as describedpreviously (Singh et al., 2012). Briefly, a single colony ofCryptococcus strains was grown in 15 ml of YPD broth at 30° C. for 20 hat 250 rpm. Cryptococcus cells (5×10⁸) were placed in a single glasstube to which the Mandala extraction buffer was added (Mandala et al.,1995). Lipid extraction was performed according to the methods of Blighand Dyer (Bligh and Dyer, 1959) followed by base hydrolysis. One set ofdried samples was resuspended in 50 μl of CHCl₃/MeOH (2:1 ratio) andanalyzed by TLC developed with chloroform/methanol/water (65:25:4,v/v/v) and stained with iodine and resorcinol, the other set was usedfor gas chromatography-mass spectrometry (GC-MS).

Lipid Profiling by Mass Spectrometry

Total lipids were extracted from Cryptococcus strains, using the methodsdescribed previously (Singh et al., 2012). For sterylglucosidesanalyses, extracted lipid samples were derivatized using N, O-bis(trimethylsilyl) trifluoroacetamide/trimethylchlorosilane reagent(Sigma-Aldrich) and then analyzed using 30 mt (0.25 μm) DB5-MS column onAgilent 7890 GC-MS (Agilent Technologies, Santa Clara, Calif., USA). Theretention time and mass spectral patterns of plant SGs standard (AvantiPolar Lipids, Inc., Alabaster, Ala., USA) were used as a reference(Gutierrez and del Rio, 2001). Cholesterol was added as an internalstandard for these analyses prior to lipid extraction. Ceramide andglucosylceramide species were analyzed by multiple reactions monitoring(MRM) as described previously (Singh et al., 2012) using TSQ QuantumUltra™ Triple Quadrupole Mass Spectrometer (Thermo Scientific, USA).Samples were delivered by Accela pump (Thermo Finnigan, USA) to the HPLCfitted with 3 μm C8SR, 150 mm×3.0 mm column (Peeke Scientific,Sommerset, N.J., USA). C17 sphingosine and C17 ceramide were added as aninternal standard for these analyses prior to lipid extraction.Determination of plant sterols and sterylglucosides for enzymaticactivity assay was performed using MRM monitoring on LC-MS (Wewer etal., 2011). Standard plant sterols and sterylglucosides (Avanti PolarLipids, Inc.) were used as the external standards in these measurements.

Animal Studies

Four weeks old female CBA/J mice (Harlan Laboratories, Indianapolis,Ind., USA) were used for all studies. Mice were anesthetized with anintraperitoneal injection of 60 μl xylazine/ketamine mixture containing95 mg ketamine and 5 mg xylazine per kilogram of body weight andinfected. For the infection studies, 24 mice (eight for each group) wereinfected intranasally with 5×10⁵ cells/20 μl of WT, Δsgl1 or Δsgl1+SGL1reconstituted strain. Mice were inspected twice a day and those thatappeared moribund or in pain were sacrificed with CO₂ inhalationfollowed by cervical dislocation. All animal procedures were approved byStony Brook University Institutional Animal Care and Use Committee andfollowed the guidelines of American Veterinary Medical Association. Fortissue burden analysis, four mice per strain were used. Lung, brain,liver, kidney and spleen were excised and homogenized in 10 ml of PBSusing Stomacher 80 (Seward, UK) for 2 min at high speed. Severaldilutions were plated in duplicate onto YPD agar plates and incubatedfor 48-72 h at 30° C. The CFUs per organ were counted. Forhistopathology analysis, three mice per strain were used. Mice organswere fixed in 3.7% of formaldehyde in paraffin and stained withhaematoxylin and eosin and mucicarmine.

For in vivo vaccination studies, mice were pre-treated with vehicle(PBS), Δsgl1 (5×10⁵ cells), and Δgcs1 (5×10⁵ cells). After 30 days, micepre-treated with vehicle or Δsgl1 were challenged with 5×10⁵ cells of CnWT or Cg R265. Mice pre-treated with Δgcs1 were challenged with 5×10⁵cells of Cn WT. Mouse survival was monitored for 80 days afterpost-challenge. CD4⁺ T-cell depletion was achieved by weeklyintraperitoneal administration of anti-CD4⁺ (GK1.5, rat IgG2b, 200 μg in200 μL of PBS; National Cell Culture Center, Minneapolis, Minn., USA). Arat IgG2b (eBioscience, Inc., San Diego, Calif., USA) was used ascontrol. T-cell depletion was assessed by flow cytometry in the spleens.For vaccination studies, mice (eight for each group) were pre-treatedwith vehicle (PBS) or Δsgl1 strain after 48 h from the first round ofT-cell depletion and after 30 days were challenged with a lethal dose ofCn WT (5×10⁵ cells) and their survival was monitored for 80 days.

Example 1: CNAG_05607 has Sterylglucosidase and not GlucosylceramidaseActivity

An S. cerevisiae expression system was used for characterizing theactivity of the CNAG_05607 enzyme. The blast search of CNAG_05607 inSaccharomyces genome database revealed a gene YIR007W with an identityof 41% (expect=1.8e-129) to CNAG_05607 (FIG. 5). Therefore, a ScΔYIRmutant strain lacking of YIR007W gene was used for the studies.CNAG_05607 was cloned in pYES/CT vector and overexpressed in S.cerevisiae YIR007W mutant strain (ScΔYIR+Cn 5607). As a negativecontrol, ScΔYIR mutant was transformed with pYES/CT empty vector(ScΔYIR+empty vector). Total proteins were extracted from S. cerevisiaestrains, which contained the empty vector (control) or overexpressed theCNAG_05607 enzyme, and were incubated with plant (FIG. 1A) orcryptococcal sterylglucosides (SGs; FIG. 1B). With either substrate, 100μg of total protein extract was enough to significantly degrade the SGsas evidenced by the disappearance of the SGs band on the TLC. Nodifference in the intensity of the SGs band was detected compared to theSG control when ScΔYIR mutant strain carrying the empty vector wasincubated with plant or cryptococcal SGs. The activity of the enzyme wasdependent on pH (FIG. 1C) and temperature (data not shown), with themaximum activity observed at a pH 4.5 in sodium acetate buffer and atemperature of 37° C. In addition to cryptococcal SGs, the CNAG_05607enzyme was also able to degrade cholesterol glucoside, the mammalianform of SGs (FIG. 6).

The CNAG_05607 enzyme has recently been characterized as aglucosylceramidase due to its ability to hydrolyze short-chainglucosylceramides (Watanabe et al., 2015). Our initial biochemicalcharacterization also showed that CNAG_05607 metabolizes short chainglucosylceramide (data not shown) similarly to what was observed byWatanabe et al. (2015). However, CNAG_05607 did not metabolizelong-chain, physiologically relevant, Δ8-C9 methyl glucosylceramides(FIGS. 1D,E), which is the form of glucosylceramide found in C.neoformans. To the best of our knowledge, glucosylceramide synthase andglucosylcerebrosidase, do not need a co-factor or activator to exerttheir activity on long chain GlcCer (i.e., C16 GlcCer; Akiyama et al.,2013). Cerezyme, a human recombinant glucosylcerebrosidase, was used ascontrol. This enzyme metabolized NBD-C₆-GlcCer (data not shown) as wellas long-chain CryptococcusΔ8-C9 methyl glucosylceramides resulting inceramide production (FIG. 1E). Cerezyme did not exhibit activity onplants or cryptococcal SGs (data not shown). Thus, these resultsdemonstrate that CNAG_05607 has specific activity towardsterylglucosides, therefore this enzyme was re-named Sterylglucosidase 1(Sgl1).

Example 2: Deletion of SGL1 Causes Accumulation of SGs and not GlcCer

Since the Sgl1 enzyme acts to metabolize cryptococcal SGs, deletion ofthis enzyme in C. neoformans should lead to a SGs accumulating strain.This hypothesis was tested by genetically eliminating thesterylglucosidase enzyme (FIG. 7) in C. neoformans and monitoring thelipid profile by performing TLC and GC-MC on the total lipids extractedfrom the WT and the mutant strain. It was found that while the WT C.neoformans produces very little SGs, genetic elimination ofsterylglucosidase (the Δsgl1 mutant) leads to a dramatic SGsaccumulation; a phenomenon that is restored in the reconstituted strain(Δsgl1+SGL1; FIGS. 2A,B). In agreement with the in vitro activitystudies, elimination of sterylglucosidase did not affectglucosylceramide levels in the cell (FIGS. 2E-2F), further confirmingthe sterylglucosides-specific activity of this enzyme.

In depth analysis of the MS spectrum of Δsgl1 strain showed theaccumulation of 9 structures (FIGS. 2B-2D) with ion fragments of m/z147, 204, 217, 305, 361, 451. These structures were characteristic oftetramethylsilyl (TMSi) glucose ion fragments resulting from cleavage ofC—O bonds. The fragments with m/z 361 and 451 are representative of TMSiderivative of hexoses. Ion fragments with m/z 129 and 255,characteristic of steroid moiety, were also present. Ion fragments withm/z of 73 and 147 represent the cleavage of 1 TMSi and 2 TMSsi groupsrespectively. The signal intensity of ion m/z 204 was greater than 217,which represented pyranoside configuration of the O-glycosidic linkage.These ion fragments resembled the fragmentation pattern generated duringthe MS analysis of plant sterylglucosides. Gutierrez and del Rio, 2001).Altogether, the ion fragments analysis confirmed that the structurepossessed all characteristics of sterylglucosides. One of the mostaccumulated structures in the Δsgl1 mutant was ergosterolglucoside (Peak2, FIG. 2C). Apart from other characteristic ion fragments ofsterolglucoside, MS fragmentation of peak 2 showed an ion fragment ofm/z 378, which results from the cleavage C—O linkage of O-linked glucosemoiety and is characteristic to ergosterol, suggesting thatergosterolglucoside was the structure with the highest concentration inthe Δsgl1 strain. The chemical structure and the electron-impact massspectrum of this molecule is presented in FIG. 2H.

Example 3: Sgl1 is a Virulence Factor of C. neoformans

Alterations in sphingolipid metabolism have been shown to attenuatecryptococcal virulence (Rittershaus et al., 2006; Singh et al., 2012).Thus, the virulence of the Δsgl1 strain in the mouse model ofcryptococcosis was tested. Mice were infected with a lethal dose offungal cells (5×10⁵ cells) to establish cryptococcosis and monitored fortheir survival. The average survival of mice infected with the WT C.neoformans was 24±6 days whereas all mice infected with Δsgl1 strainremained alive during the course of the experiment (i.e., 90 dayspost-infection). Mice infected with the Δsgl1+SGL1 strain showed asurvival pattern similar to that observed in the WT (average survival of21±7 days; FIG. 3A). During the course of infection, lungs and brainswere removed from the mice infected with the three strains and analysisof tissue burden was performed at days 0, 3, 6, 9, and 14post-infection.

Interestingly, the number of Δsgl1 cells in the lungs decreased startingat day 3 and continued until day 14, at which point the lungs werecompletely clear of fungal cells (FIG. 3B). Furthermore, no Δsgl1 cellswere observed in the brain (FIG. 3C), suggesting that fungal cells didnot disseminate to the brain in the Δsgl1-infected mice. In contrast, asignificant number of fungal cells were found in the lungs and brains ofmice infected with the WT or the Δsgl1+SGL1 strain (FIGS. 3B, C). Inboth cases, the number of fungal cells in the brain increased as afunction of time, demonstrating the occurrence of extrapulmonarydissemination and progression of the disease. The findings of the tissueburden studies were confirmed by lung and brain histology observations,which showed no fungal cells in the organs isolated from theΔsgl1-infected mice at the end of the experiment, but significant tissuedamage and presence of fungal cells in the WT or Δsgl1+SGL1 strains(FIG. 8A-8F). These experiments reveal that sterylglucosidase is avirulence factor in C. neoformans, as the loss of this enzyme leads toloss of virulence in the mouse model.

Example 4: The ΔSgl1 Strain Acts as a Vaccine Against Cryptococcosis inthe Mouse Model

Cryptococcus neoformans cells possess a number of virulence factors thatcontribute to their survival inside the host, resistance to immuneresponse, and detrimental activity against the host (Coelho et al.,2014). To gain more insight into the loss of virulence of the Δsgl1strain, a number of virulence factors in this strain were evaluated andcompared to the WT. In comparison to the WT, the Δsgl1 strain showedsimilar growth in acidic and neutral pH (at 37° C. and in the presenceof 5% CO₂), similar melanin production and capsule thickness, and nomajor difference in growth under oxidative or nitrosative stress. Inaddition, the WT and mutant strains showed similar intracellular growthduring in vitro infection of the J774.16 macrophage-like cells (FIG. 9Aand FIG. 9B). These analyses suggest that the most common virulencefactors are similar between the Δsgl1 strain and the WT denoting adifferent mechanism for the loss of virulence.

Given that the Δsgl1 strain was non-pathogenic and that SGs are knownimmunostimulators (Lee et al., 2007; Grille et al., 2010), the potentialuse of the Δsgl1 strain as a vaccine against cryptococcosis wasinvestigated. Two controls were used for these studies: a vehicle(sterile PBS) and the C. neoformans Δgcs1 strain (Rittershaus et al.,2006), which is avirulent, but does not accumulate SGs. Mice wereinfected with the vehicle, or 5×10⁵ cells of the Δgcs1 or the Δsgl1strains and after 30 days were challenged with a lethal dose of thevirulent WT C. neoformans or C. gattii R265 strains. Interestingly, themice that were pre-treated with the Δsgl1 strain were completelyprotected against the subsequent infection. However, the mice that werepre-treated with the vehicle or the Δgcs1 strain succumbed to infectionwithin 35 days (FIG. 4A). These results suggest that the Δsgl1 strainmay stimulate a host immune response that successfully kills Δsgl1 andmakes the host resistant to subsequent cryptococcosis.

Although Cryptococcus infections can afflict immunocompetentindividuals, the majority of the population at risk, are those sufferingfrom immune suppression, such as HIV/AIDS patients. A reduction in CD4⁺T-cells in this population results in aggressive cryptococcosis, whichcan be life threatening (Jarvis et al., 2013). The efficiency of theΔsgl1 strain as a vaccine against cryptococcosis during immunesuppression was examined by its administration in CD4⁺ T-cells depletedmice prior to infection with the WT C. neoformans. For these studies,mice were depleted of CD4⁺ by weekly administration of anti-CD4⁺antibody or control antibody (rat IgG2b) starting a month prior toinfection with WT C. neoformans (FIG. 4B). A 94.3% percent reduction inCD4⁺ T-cells was achieved as confirmed by flow cytometry (FIGS. 4C-4D).The Δsgl1 strain or control (PBS) was also administered to mice a monthprior to infection. Mice were then infected with 5×10⁵ cells of thevirulent WT C. neoformans. All mice that received the PBS and theantibody control succumbed to infection in 41 days, while all the CD4⁺T-cells depleted mice that were vaccinated with the Δsgl1 strainsurvived the infection, demonstrating that this strain is not infectiousand can protect immune suppressed mice against a subsequent cryptococcalinfection (FIG. 4B).

Example 5: ΔSgl1 Strain can be Used to Produce Sterylglucosides

Cryptococcus neoformans (Cn) cells produce sterylglucosides (SGs); but,in wild type fungal cells the level of this lipid in Cn cells is almostundetectable. This suggests that under normal growing conditions, wildtype Cryptococcus neoformans cells highly regulate and breakdown thislipid, and hence only very little amounts are accumulated.

It has been demonstrated herein by the present inventors that the Sgl1gene in Cryptococcus neoformans produces an enzyme that breaks downsterylglucosides. Inactivation of the sgl1 gene (or its homologs inother fungal species) leads to a significant accumulation SGs in fungalcells. The present disclosure, therefore, comprises the use of mutantfungi comprising an inactivated Sterylglucosidase (Sgl1) gene homolog toproduce SGs. The present disclosure also comprises methods of SGpurification from said mutant fungi.

An example method (protocol) for SG purification is described below.This example method (protocol) is by no means limiting, and one skilledin the art would know how to modify and optimize the given protocolfurther, e.g., for scaling up or scaling down the purification.

Briefly, mutant fungal strains are grown in 1.5 liters of YPD medium at37 C for 24 hours (exponential phase—cell concentration in this phase is˜10⁶/ml). YPD medium comprises 2% Bacto peptone (Difco), 1% Bacto YeastExtract (Difco), and 2% glucose. The grown fungal cells are counted andMandala extraction is done on the entire culture of approximately 5×10⁹total cells. Mandala extraction is carried out as described in Mandalaet al., (1995), The Journal of Antibiotics 48.5: 349-356, the entirecontents of which is expressly incorporated herein by reference. Then,lipid extraction is achieved with a Bligh and Dyer extraction on thedried Mandala tubes. Bligh and Dyer extraction is performed as describedin Bligh, E. G. and Dyer, W. J. Can. J. Biochem. Physiol., 1959,37:911-917, the entire contents of which is expressly incorporatedherein by reference and the resulting sample is dried. All the driedBligh and Dyer tubes are combined into one tube by dissolving in 4 mL ofChloroform:acetic acid (99:1). If this solution is turbid, the tube iscentrifuged for 5 min at 1500 g and supernatant is used for the column.In the next step a Sep-Pak Cis cartridge column (Sep-Pak™, WatersAssociates, Milford, Mass., U.S.A.), is washed with 90 mL chloroform.The sample from the previous step in 4 mL of Chloroform:acetic acid(99:1) is added to the wetted Sep-Pak column. Column is rinsed withanother 6 mL of Chloroform:acetic acid (99:1). The column is washed with60 mL of Chloroform:acetic acid (99:1). The column is eluted with 60 mLacetone and the flow through is collected in test tubes, which containthe sample. All sample in the test tubes from the previous step is driedin speedVac and combined in one tube, by resuspending the dried samplesin acetone and adding them to other tubes. Next, base hydrolysis isperformed on the samples in the tube as described in (Mandala et al.,1995). The sample is dried again upon the base hydrolysis step and laterdissolved in 4 mL of Chloroform:acetic acid (99:1).

Following the first column purification, a second column purification isperformed using a second Sep-Pak column. Briefly, the second column isfirst washed with 90 mL chloroform. Then the 4 mL of sample is addedslowly to the second column. The sample tube is rinsed with 6 mL ofchloroform:acetic acid (99:1) and added to the second column as well.The flow through is collected in tube 1. 20 mL of chloroform:acetic acid(99:1) is next added to the second column and collected in tube 2. 20 mLof chloroform:acetic acid (99:1) to the second column and collect intube 3. Next, 14 mL of chloroform:acetic acid (99:1) is added to thesecond column and collected in tube 4, and when chloroform:acetic acid(99:1) has flown through, a 6 mL of chloroform:methanol (95:5) is addedand collected in tube 4. 20 mL of chloroform:methanol (95:5) is added tothe second column and collected in tube 5. 14 mL of chloroform:methanol(95:5) is added to the column and collected in tube 6, whenchloroform:methanol (95:5) has flown through, then 6 mL ofchloroform:methanol (90:10) is added and collected in tube 6. 20 mL ofchloroform:methanol (90:10) is added to the second column and collectedin tube 7. Another 20 mL of chloroform:methanol (90:10) is added to thecolumn and collected in tube 8. Next, 14 mL of chloroform:methanol(90:10) is added to the column and collect in tube 9. Finally, 6 mLmethanol is added to the second column and collected in tube 9. Alltubes (1-9) are dried in the SpeedVac and then all lipids are dissolvedin 1 mL of chloroform:methanol 2:1. For quality control 100 uL (10% ofthe overall sample) is dried it in SpeedVac, redisperses in 15 uL ofChloroform:methanol (2:1) and run on TLC (Thin layer Chromatography)plates to make sure that the purified lipid is found in tube 7. Whenrunning the TLC a plant sterolglycoside standard is run on the side ofthe sample in tube 6, to make sure that the SGs are properly purified.The TLC looks as shown in FIG. 10.

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What is claimed is:
 1. A composition comprising a mutant fungus, whereinsaid mutant fungus comprises an inactivated Sterylglucosidase (Sgl1)gene or homolog thereof.
 2. The composition of claim 1, wherein saidmutant fungus lacks the ability to metabolize sterylglucosides (SGs). 3.The composition of claim 1, wherein said mutant fungus accumulatessterol glycosides.
 4. The composition of claim 1, wherein said mutantfungus is avirulent.
 5. The composition of claim 1, wherein said mutantfungus is from a Cryptococcus genus.
 6. The composition of claim 1,wherein said mutant fungus is selected from the group consisting ofCryptococcus neoformans, Cryptococcus gatii, Cryptococcus albidus,Cryptococcus uniguttulatus, Candida albicans, Aspergillus fumigatus andother fungi in which the Sgl1 gene is deleted.
 7. A method for producingsterylglucosides comprising: providing a mutant fungus comprising aninactivated Sterylglucosidase (Sgl1) gene or homolog thereof; expressingsaid mutant fungus in a fungal cell, wherein said fungal cell producessterylglucosides; and isolating said sterylgucosides.
 8. The method ofclaim 7, wherein said mutant fungus lacks the ability to metabolizesterylglucosides (SGs).
 9. The method of claim 7, wherein said mutantfungus accumulates sterol glycosides.
 10. The method of claim 7, whereinsaid mutant fungus is from a Cryptococcus genus.
 11. The method of claim7, wherein said mutant fungus is selected from the group consisting ofCryptococcus neoformans, Cryptococcus gatii, Cryptococcus albidus,Cryptococcus uniguttulatus, Candida albicans, Aspergillus fumigatus andother fungi in which the Sgl1 gene is deleted.